A critical component in the defense against viruses and tumors relies on the presentation of viral or tumor derived peptides to CD8+ T-cells in the context of major histocompatibility class I molecules (MHC I). Successful presentation of such peptide antigens elicits cell mediated immune responses specific for host cells displaying these peptide antigens and thus serves as the central mechanism for eradication of abnormal (infected or transformed) cells. One challenge in the generation of vaccines that specifically stimulate cell-mediated immune responses has been the difficulty of loading cell-surface MHC I molecules with relevant peptide antigens in vivo. Recently, approaches for the direct cell-surface loading of MHC class I molecules have emerged that are dependent on the presence of beta-2 microglobulin (b2m) in the peptide innoculum, and, as we show, are based on the ability of b2m to stabilize newly emerging empty class I MHC molecules and make them receptive to peptide loading. This "adjuvant" activity of b2m is dependent on its affinity for the MHC heavy chain, and has led to our studies in which we have engineered higher affinity variants of human b2m that appear to have improved abilities to stabilize MHC I heavy chains on the cell-surface, facilitate peptide loading, and stimulate MHC restricted, peptide specific T-cells in vitro. The high affinity variants currently being evaluated have mutations that promote either the formation of a salt bridge between residues of the MHC heavy chain and b2m , or promote hydrophobic interactions and the exclusion of water molecules from the interface between the two chains, both of which should increase the strength of association between the chains. These proteins are currently also being evaluated for their ability to stimulate cell-mediated immune responses in vivo, and will be used to study effects of MHC I presentation in thymic development. In addition, we are engineering fusion proteins capable of providing co-stimulatory signals necessary to stimulate generation of primary cell-mediated immune responses to MHC I bound peptide antigens. Our hope is that these high-affinity b2m adjuvants and co-stimulatory fusion proteins will lead to the development of novel peptide-based vaccines for the treatment of viral infections and cancers.